Facultative Heterochromatin

 Facultative heterochromatin is the term describing regions of chromosomes that appear heterochromatic at certain times in the cell cycle but are not always this way. This is contrasted with constitutive heterochromatin, which is always condensed and stains strongly throughout the cell cycle (1). Facultative heterochromatin represents domains of euchromatin that are condensed and inactive in a particular cell. An excellent example of the assembly of facultative heterochromatin is found in the inactive X-chromosome. In the inactive X-chromosome, the histones are hypoacetylated, DNA is methylated, and the chromosome replicates late in S-phase. All of these traits are characteristic of inactive chromatin. Importantly, these are not irreversible modifications and can be reversed at certain points in development.

 The various characteristics of facultative heterochromatin effectively work together to stabilize a transcriptionally repressed state. The generation of antibodies against acetylated histones has allowed a number of general correlations concerning the possible functional roles of histone acetylation. There is also a strong correlation between histone acetylation and the transcriptional activity of chromatin. In Saccharomyces cerevisiae most of the genome is transcriptionally active and contains hyperacetylated core histones. Transcriptionally inactive domains of chromatin in yeast, such as the silent mating type cassettes and telomeric sequences, contain histone H4 that is hypoacetylated, except at one position, Lys12 (2). In higher eukaryotes, acetylation of histone H4 increases during the reactivation of transcription in the initially inactive chicken erythrocyte nucleus, following fusion of the erythrocyte with a transcriptionally active cultured cell, to form a heterokaryon. Histone acetylation is particularly prevalent over the specific b-globin genes that are actively transcribed in reticulocytes. More recent studies have demonstrated convincingly that histone hyperacetylation is actually restricted to the domain of chromatin that contains the potentially active chicken b-globin gene locus . This result indicates very specific targeting of histone acetyltransferase activity. Immunolabeling of polytene chromosomes in Chironomus and Drosophila also reveals a nonrandom distribution of histone H4 acetylation that correlates with transcriptional activity. Within female mammals, the transcriptionally inactive X-chromosome is distinguished by a lack of histone H4 acetylation (3). Therefore several independent experimental approaches have shown that actively transcribed and potentially active chromatin domains are selectively enriched in hyperacetylated histones, whereas transcriptionally inactive chromatin contains hypoacetylated histones.

 A role for specialized chromatin structures in mediating transcriptional silencing by methylated DNA, like that found in the facultative heterochromatin of vertebrates, has been suggested by several investigators. High levels of methyl-CpG correlate with transcriptional inactivity and nuclease resistance in vertebrate chromosomes (4). Methylated DNA transfected into mammalian cells is also assembled into nuclease-resistant structures within the assembled minichromosomes, indicating the existence of unusual nucleosomal particles (5). These unusual nucleosomes migrate as large nucleoprotein complexes in agarose gel electrophoresis. These complexes are held together by higher order protein–DNA interactions, despite the presence of abundant micrococcal nuclease cleavage points within the DNA. Individual nucleosomes assembled on methylated DNA interact together more stably than on unmethylated templates.

 The accessibility of chromatin to nucleases could also be affected directly by the stability with which the histones interact with DNA within the nucleosome. DNA methylation does not influence the association of core histones with the vast majority of DNA sequences in the genome. However, for certain specific sequences, such as those found in the Fragile X mental retardation gene 1 promoter, methylation of CpG dinucleotides alters the positioning of histone–DNA contacts and the affinity with which these histones bind to DNA. The exact chromatin structure found in vivo can also result from gene activity. Linker histones, such as H1, are relatively deficient in the transcribed regions of genes. So it is not surprising that transcriptionally inactive facultative heterochromatin containing methyl-CpG should show an increase in the abundance of histone H1, whereas DNA sequences lacking methyl-CpG are deficient in H1. In vitro studies indicate that histone H1 interacts preferentially with methylated DNA under certain conditions.

 There are also features of transcriptional repression dependent on methylated DNA that can be explained by methylation-specific repressors that operate more effectively within a facultative heterochromatin environment. Transcriptional repression is strongly related to the density of DNA methylation. A non linear relationship exists between the lack of repression observed at low densities of methyl CpG and repression at higher densities. These results led to the demonstration that local domains of high methyl-CpG density confer transcriptional repression on unmethylated promoters in cis. The importance of a nucleosomal infrastructure for transcriptional repression dependent on DNA methylation is shown by the observation that immediately after injection into Xenopus oocyte nuclei, methylated and unmethylated templates both have equivalent activity (6). As facultative heterochromatin is assembled, however, the methylated DNA is repressed with the loss of DNase I hypersensitive sites and the loss of engaged RNA polymerase . The requirement that nucleosomes exert efficient transcriptional repression dependent on DNA methylation can be explained in several ways. Methylation-specific repressors might recruit a corepressor complex that directs the modification of the chromatin template into a more stable and transcriptionally inert state. One potential candidate corepressor for MeCP2 is the SIN3-histone deacetylase complex, because inhibition of histone deacetylation reverses some of the transcriptional repression conferred by DNA methylation (7). Alternatively, like histone H1, methylation-specific repressors might bind more efficiently to nucleosomal rather than to naked DNA. Any cooperative interactions between molecules could propagate their association of methylation-specific repressors along the nucleosomal array even into unmethylated DNA segments. This latter mechanism is analogous to the nucleation of heterochromatin assembly at the yeast telomeres by the DNA-binding protein RAP1, which then recruits the repressors SIR3p and SIR4p that organize chromatin into a repressive structure (8). All of these potential mechanisms could individually or together contribute to assembling of a repressive facultative heterochromatin domain.

A final relevant issue is the significance of the timing of replicative initiation on facultative heterochromatin in S-phase. Facultative heterochromatin is normally replicated late in the S-phase. If replication disrupts both active and repressed chromatin structures, then the entire nucleus has to be remodeled after each replication. The accessibility of immature chromatin on newly replicated DNA provides a means to accomplish this remodeling. The reformation of nuclear structures, however, has other implications. If the transcription factors available in a cell are limiting, then a gene that is replicated early in the S-phase has more opportunity to assemble an active transcription complex than a gene that replicates late simply because the gene that replicates early is available for transcription factors to bind to it before all of the early replicating portion of the genome has sequestered these factors. Therefore a late-replicating gene in facultative heterochromatin experiences a relative deficiency in transcription factor availability. Transcriptionally active genes in euchromatin replicate early in the S-phase. The reason for this early replication is unknown, but possibilities include the local disruption of chromatin structure by transcription complexes, which makes that DNA more accessible to the replication machinery (9) and the observation that many transcription factors are in fact also replicative factors. An attractive variation of this model is that the type of chromatin assembled early in the S-phase is more accessible to transcription factors than chromatin assembled late in the S-phase. Early replicating chromatin may sequester histones that are more highly acetylated and consequently more accessible to the transcription factors that maintain continued transcriptional activity. The CENP-A protein that replaces histone H3 in the mammalian centromere within heterochromatin is synthesized at the very end of the S-phase, providing an example of how the cell cycle-dependent compartmentalization of protein biosynthesis might contribute to assembling a specialized chromatin structure (10).  Although a general test of the significance of this model has not been made, it remains an attractive mechanism for explaining both the maintenance of specific patterns of gene expression in a proliferating cell type and the maintenance of domains of facultative heterochromatin.

References

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